ZDC (Zika, Dengue,

and Chikungunya)
Multiplex RT-PCR Assay
Instruction Manual

RT-PCR assay for the detection of Zika, dengue,

and chikungunya viral RNAs

Catalog #12003818

For research use only.

ii  |  ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay
Table of Contents

Section 1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Section 2 Equipment and Consumables. . . . . . . . . . . . . . . . . . . . . . . . . . 5

Section 3 Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Section 4 Extraction of Nucleic Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Section 5 RT-PCR Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Section 6 Interpretation of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Section 7 Assay Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay  |  iii

Section 1 Introduction

Section 1
Introduction
Name and Intended Use
The ZDC Multiplex RT-PCR Assay is a real-time PCR test intended for the
detection of RNA from Zika virus (ZIKV), dengue virus (DENV), and chikungunya
virus (CHIKV). The results of the assay determine whether ZIKV, DENV (DEN-1,
DEN-2, DEN-3, and DEN-4), and CHIKV viral RNA was detected.

The ZDC Multiplex RT-PCR Assay is intended for research use only and not for
use in diagnostic procedures. The assay has been wet-lab validated for use
with Bio-Rad’s CFX96™ and CFX96 Touch™ Real-Time PCR Detection Systems
and Thermo Fisher Scientific’s Applied Biosystems 7500 Fast Dx Real-Time
PCR System. The assay is also compatible with other commonly used real-
time PCR instruments (see Table 1 on page 9 for system compatibility).

Protocol Use Limitations

The ZDC Multiplex RT-PCR Assay is intended for research use only and must
not be used for diagnostic purposes.

Summary and Explanation of the Test

The ZDC Multiplex RT-PCR Assay is a real-time reverse transcription polymerase
chain reaction (RT-PCR) test that enables the detection of ZIKV, DENV (DEN-1,
DEN-2, DEN-3, and DEN-4 can be detected but the specific strain cannot be
identified), and CHIKV viral RNAs in a variety of sample types.
Purified nucleic acids are reverse transcribed and amplified in a single step
that is carried out without any extra pipetting from the operator.* The reverse
transcription step takes advantage of the iScript™ Reverse Transcriptase
(Bio-Rad Laboratories, included in kit) and the resulting cDNA is subsequently
amplified using the iTaq™ Universal Probes One-Step Reaction Mix (Bio-Rad
Laboratories, included in the kit). The reverse transcription/amplification reaction
can be performed on commonly available real-time PCR systems (see Table 1
on page 9 for system compatibility).
The assay takes advantage of specific primers to first reverse transcribe RNA
into cDNA and then amplify the target cDNA. The ZIKV RT-PCR assay targets
the ns4b gene, the DENV assay targets 5'UTR sequences, and the CHIKV
assay targets the nsP2 gene. During amplification, the probes bind to the

* It is responsibility of the researcher to select and use the most appropriate methodology to obtain
PCR-grade viral RNA from the sample type of interest.

1  |  ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay

 Section 1 Introduction

ZIKV, DENV, and CHIKV target sequences. Subsequent extension of the PCR
product leads to hydrolysis of the probe and separates the probe’s fluorescent
reporter from the quencher molecule. Subsequently, the presence of target
cDNA is determined in real time by the detection of target-specific fluorescent
oligonucleotide probes during amplification. A multiplexed PCR probe mix allows
simultaneous amplification and detection of all three viruses (when present) and
an internal control in a single reaction. In the absence of target RNA/cDNA for a
given oligonucleotide probe, no corresponding fluorescence will be emitted; thus
no signal will be detected.
Optional RNase P assays specifically designed for maximum compatibility with
the ZDC Multiplex RT-PCR Assay are available with Cy5.5 (compatible with the
CFX96, CFX96 Touch, and other qPCR systems) and TAMRA labeling and can
be purchased separately. See Table 1 on page 9 for system compatibility. An
RNase P positive control, ZDC Clear, is also available and can be purchased
separately (catalog #12004860).

Safety/Precautions
Good laboratory practice is compulsory and strict universal safety precautions
must be taken for all activities that require the handling of samples/specimens
that can be biohazardous, infectious, or contain pathogens.
The CDC provides biosafety guidance for working with Zika virus specimens at
cdc.gov/zika/state-lab/index.html. Risk assessment when conducting a new
laboratory test is recommended and safety precautions should be based on
the outcome of the laboratory’s risk assessment.
While Zika and dengue viruses have to be handled under a biosafety level 2
(BSL-2) environment, specimens that potentially harbor chikungunya virus
should be handled in a BSL-3 laboratory.*
Always consider the potential danger of the specimens being tested while
performing all procedures described in this document and/or the workflow
for this assay. The assay has been designed to identify the presence of viral
genomes. Therefore, it is assumed that the specimens contain viruses that
are considered biohazardous.
Operators should be aware of the risks and safety procedures and should
recognize that chikungunya produces high levels of viremia and that
specimens from suspected chikungunya virus cases should be treated
as potentially infectious.

* Visit cdc.gov/zika/state-lab/index.html to find CDC guidelines for state and local public health
laboratories. For additional information, visit cdc.gov/biosafety/publications/bmbl5/index.htm for
Biosafety in Microbiological and Biomedical Laboratories BMBL guidelines.

ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay  |  2

Section 1 Introduction

General safety procedures and precautions

■■  his test kit should be handled only by qualified personnel trained in
T
laboratory procedures and real-time PCR techniques and familiar with their
potential hazards. Handle appropriately with the requisite Good Laboratory
Practices. Wear protective clothing, including lab coat, eye/face protection,
and disposable gloves (synthetic, non-latex gloves are recommended)
while handling kit reagents and patient samples. Wash hands thoroughly
after performing the test
■■  se extreme caution when handling clinical specimens as the primary
U
hazard associated with blood specimens from patients suspected of
infection with CHIKV is inhalation of virus-containing aerosols. As
coinfection with ZIKV, DENV, and CHIKV can occur, treat all specimens
as potentially infectious
■■  o not smoke, drink, or eat in areas where specimens or kit reagents
D
are being handled
■■ Do not pipet by mouth
■■  o known test method can offer complete assurance that infectious
N
agents are absent. Therefore, all human blood derivatives, reagents,
and human specimens should be handled as if capable of transmitting
infectious disease. Follow recommended standard and universal
precautions for blood-borne pathogens as defined by OSHA, Biosafety
Level 2 guidelines from the current CDC/NIH Biosafety in Microbiological
and Biomedical Laboratories, the WHO Laboratory Biosafety Manual,
and/or local, regional, and national regulations
■■ 
Biological spills: human source material spills should be treated as
potentially infectious
■■ Spills not containing acid should be immediately decontaminated, including
the spill area, materials, and any contaminated surfaces or equipment,
with an appropriate chemical disinfectant that is effective for the potential
biohazards of the samples involved (1:10 dilution of household bleach,
70–80% ethanol or isopropanol, an iodophor such as 0.5% Wescodyne
Plus, EPA registration #4959-16-52, or a phenolic) and wiped dry
■■  pills containing acid should be appropriately absorbed (wiped up) or
S
neutralized and wiped dry. The area should be decontaminated with
a chemical disinfectant. Materials used to absorb the spill may require
biohazardous waste disposal
NOTE: DO NOT PLACE SOLUTIONS CONTAINING BLEACH INTO
THE AUTOCLAVE

3  |  ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay

 Section 1 Introduction

■■  ispose of all specimens and material used to perform the test as though
D
they contain an infectious agent. Laboratory chemical or biohazardous
wastes must be handled and discarded in accordance with all local,
regional, and national regulations
■■  omplete hazard information and precautions are located in the Safety
C
Data Sheet (SDS) available from Bio-Rad Technical Service and at
bio-rad.com

ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay  |  4

Section 2 Equipment and Consumables

Section 2
Equipment and Consumables
Components Included in the ZDC Multiplex RT-PCR Assay
ZDC Multiplex RT-PCR Assay - Catalog #12003818 (200 tests):

■■ iTaq™ Universal Probes One-Step Reaction Mix, 2x, 3 x 1 mL /tube

■■ iScript™ Reverse Transcriptase, 5x, 1 x 100 μL
■■ ZDC Multiplex PCR Assay Mix, 12.5x, 1 x 440 μL
■■  DC Internal Control, lyophilized (resuspend in 1 mL ZDC Buffer T and
Z
dilute 1:100), 1 tube
■■  DC Positive Control, lyophilized (resuspend in 200 μL ZDC Buffer T),
Z
1 tube
■■ ZDC Buffer T, 4 x 2 mL /tube
■■ Nuclease-free water, 2 x 800 μL

Note: The ZDC Multiplex RT-PCR Assay includes enough ZDC Buffer T to run
approximately 28 positive controls. Additional ZDC Buffer T can be purchased
separately, if needed (catalog #12005062).

Equipment Required and Not Provided in the ZDC Multiplex

RT-PCR Assay
■■  eal-time qPCR instrument (CFX96™ or CFX96 Touch™ Real-Time PCR
R
Detection System (recommended) or other compatible qPCR systems.
See Table 1 on page 9 for system compatibility.)
■■ Vortex mixer
■■ Microcentrifuge
■■ Appropriate containers to prepare reagents
■■ Precision pipets to deliver 0.5 μL to 1 mL of sample
(accurate within ±10%) or a multichannel pipettor

Consumables Required but Not Provided in the ZDC

Multiplex RT-PCR Assay
■■ 1.5 mL microcentrifuge tubes for RNA sample collection
■■  CR tubes and caps or PCR plates (suggested plates for the CFX96
P
Systems: catalog #HSP9655)
■■ Racks for microcentrifuge tubes
■■ Pipet tips, sterile, nuclease-free, and aerosol resistant
5  |  ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay
 Section 2 Equipment and Consumables

■■ Appropriate containers to prepare reagents

■■  ousehold bleach (5% to 8% sodium hypochlorite), which may be
H
diluted to a minimum concentration of 10% bleach (or 0.5% sodium
hypochlorite), or other RNA/DNA surface decontaminants
■■ Disposable gloves
■■ Molecular-grade water, glass distilled, RNase free

Storage and Stability

Store the kit at –20°C. The assay is stable for six months at this temperature.
Bring all reagents to 2–8°C before use. Return all reagents to –20°C
immediately after use.

Instrument Compatibility
The ZDC Multiplex RT-PCR Assay has been wet-lab validated on Bio-Rad’s
CFX96 and CFX96 Touch Real-Time PCR Systems and Thermo Fisher
Scientific’s Applied Biosystems 7500 Fast DX Real-Time PCR System. The
assay is also compatible with other commonly used real-time PCR systems
(Table 1). If your system is not listed, please contact Bio-Rad Technical Support
in your region to check for potential compatibility. The user should select the
setup appropriate for their PCR system. More details are available under
PCR Instrument Programming — Fluorescence Detectors (Dyes)
on page 17.
Table 1. Noncomprehensive list of real-time PCR systems compatible with the
ZDC Multiplex RT-PCR Assay.
4-Plex 5-Plex
Capability Capability (ZIKV, RNase P
(ZIKV, DENV, DENV, CHIKV, Compatible
Real-Time PCR CHIKV, Internal Internal Control, Assay, ROX
Vendor Instrument Control) RNase P) Catalog # Normalization
Bio-Rad CFX96 and Yes Yes 12004601 Not required
CFX96 Touch
Thermo Applied Yes Yes 12004602 Not required
Fisher Biosystems 7500
Scientific QuantStudio 5 Yes Yes 12004601 Not required
or 12004602
QuantStudio 6 Yes Yes 12004602 Not required
Flex
QuantStudio 7 Yes Yes 12004601 Not required
Flex or 12004602
QuantStudio 12K Yes Yes 12004601 Not required
Flex or 12004602
QIAGEN Rotor-Gene Yes Yes 12004601 Not required
Roche LightCycler 480 Yes Yes 12004601 Not required
or 12004602
LightCycler 96 Yes No N/A Not required

ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay  |  6

Section 2 Equipment and Consumables

Precautions for Users

1. Do not use the kit beyond the expiration date.
2. Do not use reagents from other manufacturers with this assay.
3. Use disposable DNase-/RNase-free pipet tips that are aerosol resistant.
4. A
 void nuclease (DNase/RNase) and microbial contamination of the
specimen and components of the kit.
5. U
 se segregated working areas for (i) specimen preparation, (ii) reaction
setup, and (iii) amplification/detection activities. Use dedicated supplies and
equipment in each area. Workflow in the laboratory should always proceed
in the same direction.
6. A
 lways wear disposable gloves and change them before entering
different areas.
7. T
 o avoid contamination with amplicons, do not open the reaction tubes/
plates after amplification.
8. S
 tore positive and/or potentially positive material separately from all other
kit components.

Reagent Preparation and Storage

Stock Reagent Preparation and Handling
Every time reagents need to be prepared, clean all working surfaces with
10% bleach followed by reagent alcohol.

Preparing the stock internal control (IC) RNA solution

1. Resuspend the lyophilized IC RNA with 1 mL of ZDC Buffer T.
2. D
 ilute the resuspended IC RNA to 1:100 by combining 1 µL of
resuspended IC RNA with 99 µL of ZDC Buffer T.
3. D
 ivide the 1:100 diluted IC RNA into aliquots and label with preparation
date, lot number, and expiration date. Store at –20ºC for future use. Do
not freeze-thaw IC RNA more than three times.

The IC RNA solution should be added to the lysis/extraction buffer or directly

into the RNA sample after resuspension into the lysis buffer. The volume of
IC RNA solution that should be added depends on the elution volume and should
be 1/60 of the elution volume (for example, 1 µL in 60 µL total elution volume).

7  |  ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay

 Section 2 Equipment and Consumables

Preparing the stock positive control RNA solution

1. R
 esuspend the lyophilized positive control RNA with 200 µL of ZDC
Buffer T.
2. D
 ivide the resuspended positive control RNA into aliquots and label with
preparation date, lot number, and expiration date. Store at –20ºC for future
use. Do not freeze-thaw positive control RNA.

Negative- and Positive-Sample Control Preparation

Preparing the negative-sample control
To prepare the negative-sample control, add 1 µL of stock IC RNA solution
from the aliquoted sample (step 3, Preparing the stock internal control (IC)
RNA solution on page 10) to 140 μL of ZDC Buffer T. The resulting solution
is the negative-sample control and should be extracted in parallel with the
experimental samples and other controls.

Preparing the positive-sample control

To prepare the positive-sample control, add 1 µL of stock positive control RNA
solution from the aliquoted sample (step 2, Preparing the stock positive
control RNA solution on page 11) to 140 µL of ZDC Buffer T. The resulting
solution is the positive-sample control and should be extracted in parallel with
the experimental samples and other controls.

ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay  |  8

Section 3 Quality Control

Section 3
Quality Control
Real-time RT-PCR is a sensitive procedure and should be conducted
following strict quality control and quality assurance procedures. Following
these guidelines will help minimize the chance of false-positive and false-
negative results.

General Considerations
■■  perators assigned to use this assay should be trained professionals and
O
familiar with the protocol and instruments used
■■  se segregated working areas for (i) specimen preparation, (ii) reaction
U
setup, and (iii) amplification/detection activities. Use dedicated supplies and
equipment in each area. Workflow in the laboratory should always proceed
in the same direction
■■  he workflow should always follow one direction from RNA extraction and
T
reagent preparation that must be performed in a clean area to RT-PCR
reaction setup that should be performed in a dirty area. This approach
will minimize the risk of contamination of the clinical samples by amplified
nucleic acids
■■  se different pairs of powder-free gloves for the work carried out in the
U
clean area and for the procedures conducted in the dirty area
■■  o not use reagents from other manufacturers with this assay (unless
D
specified in this document)
■■ Use disposable DNase-/RNase-free pipet tips that are aerosol resistant
■■  void nuclease (DNase/RNase) and microbial contamination of the
A
specimens and kit components
■■  o avoid contamination with amplicons, do not open the reaction tubes/
T
plates after amplification
■■  tore positive and/or potentially positive material separately from all other
S
kit components

Assay Control
Internal control
Bio-Rad’s ZDC Multiplex RT-PCR Assay includes a ZDC Internal Control.
The ZDC Internal Control is composed of a known synthetic RNA sequence
that has no homology with any human sequence. It is added either to the
RNA lysis/extraction buffer or to the RNA sample once resuspended into

9  |  ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay

 Section 3 Quality Control

the lysis buffer. The internal control will then be purified, reverse transcribed,
and amplified together with any viral RNA present in the sample. This control
provides information regarding the quality of the extraction and the potential
carryover of inhibitors. The primers and probe for detection of the internal
control by RT-PCR are included in the ZDC Multiplex RT-PCR Assay Mix.
The internal control should also be added to the positive controls.

Positive control
The ZDC Positive Control includes three in vitro transcribed RNA molecules
carrying sequences that are homologous to Zika (ZIKV), dengue (DEN-1)
(DENV), and chikungunya (CHIKV) viruses and are targeted by the ZDC
Multiplex RT-PCR Assay. These synthetic RNAs should be spiked into ZDC
Buffer T or ZDC Clear (catalog #12004860) as described in Section 2 —
Equipment and Consumables — Reagent Preparation and Storage. This
control should be extracted and run in parallel any time a test is conducted
and a reaction is prepared.

Note: Positive controls represent a source of cross-contamination. Precautions

should be taken to prevent and minimize the risk. To reduce the risk of cross-
contamination, refer to the General Considerations in this section.

No template control (NTC)

NTC reactions include PCR-grade water (provided with the ZDC Multiplex
RT-PCR Assay) in place of specimen RNA. This control should be included for
each reaction mixture in each run. This control provides important information
about the presence of potential contaminants and functionality of the
assay/environmental contaminations that result in false-positive results.
Negative control
The negative control is composed of internal control (IC) RNA–spiked ZDC
Buffer T (see Preparing the negative-sample control on page 11). This
control should be run in parallel with the experimental samples each time a run
is set up. It provides information about the presence of potential environmental
contaminants and the eventual malfunctioning of primers/probes.

Sample control (optional)

RNase P assays (catalog #12004601 or 12004602) can be used as sample
controls together with ZDC Clear (catalog #12004860). This control can be
included in the ZDC RT-PCR Assay as an additional target or run separately.
ZDC Clear will serve as a positive control for RNase P and a negative control
for viral targets. This control provides information about the quality of the
starting material and the extraction procedure.

ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay  |  10

Section 4 Extraction of Nucleic Acids

Section 4
Extraction of Nucleic Acids
Notes on Extraction
■■  he ZDC Internal Control should be added in each extraction run as an
T
internal control
■■  ever add carrier RNA (cRNA), internal control (IC) RNA, or positive control
N
RNA directly to the sample as enzymes potentially present in the specimen
could degrade the RNA
■■  RNA is critical to maintaining the extraction efficiency and stability of
c
extracted RNA
■■  limination of any carryover ethanol from the extraction to the PCR step
E
is crucial; ethanol will strongly inhibit real-time PCR

11  |  ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay

 Section 5 RT-PCR Procedure

Section 5
RT-PCR Assay
Preliminary Statement
1. A
 ssay controls should be run with all test samples. Assay validity
is determined by the controls run with each assay.
2. Each individual run should include three control reactions:
■■  negative control (negative for Zika (ZIKV), dengue (DENV),
1
and chikungunya (CHIKV)): ZDC Clear + internal control (IC) RNA
or ZDC Buffer T + IC RNA
■■  positive control (positive for ZIKV, DENV, and CHIKV): positive control
1
+ ZDC Buffer T
■■ 1 no template control (H2O)

RT-PCR Assay Procedure

Preparation of the PCR Master Mix
1. T
 haw and mix the reagents carefully by pipetting up and down or flicking
the tubes gently. Briefly spin reagent vials in a microcentrifuge before
opening. Store in a cooling block prior to use and limit exposure to light.
2. P
 repare the PCR master mix in a 1.5 mL microcentrifuge tube according to
Table 2. Store in a cooling block and limit exposure to light until ready to use.

Table 2. Preparation of the reaction mix.

ZDC Assay Reaction Mix 1x 10x

2x iTaq™ Universal Probes One-Step Reaction Mix, µL 12.5 125
iScript Reverse Transcriptase, µL
™
0.6 6
ZDC Multiplex PCR Assay Mix (12.5x), µL 2 20
Template, µL (5) –*

Water (molecular grade), μL 4.9 49

Total, µL 25 200
* 5 μL of sample will be added to each tube separately.

Note: Prepare 10% excess volume of master mix to ensure accurate pipetting.

ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay  |  12

Section 5 RT-PCR Procedure

Reaction Setup

1. P
 ipet 20 μL of the master mix into each well of an optical 96-well reaction
plate, as required, or into an optical reaction tube.
2. A
 dd 5 μL of sample (eluate from the nucleic acid extraction) or 5 μL of a
control (positive, negative, and NTC control). At least one positive, one
negative, and one NTC control must be included in each run.
3. S
 eal the 96-well reaction plate with an optical adhesive film or the reaction
tubes with appropriate lids.
4. T
 horoughly mix the samples and controls with the master mix by vortexing
for 15 sec.
5. C
 entrifuge the 96-well reaction plate in a centrifuge with a microtiter plate
rotor for 30 sec at approximately 1,000 x g (~3,000 rpm).

PCR Instrument Programming

Details regarding the setup and programming of Bio-Rad's CFX96™ or
CFX96 Touch™ Real-Time PCR Detection Systems or other real-time PCR
instruments can be found in the system instruction manual or by contacting the
manufacturer. Apply the following parameters for the multiplex RT-PCR assay.
1. Settings

Settings
Reaction volume 25 μL
Ramp rate Default
Passive reference None

Note: Bio-Rad's CFX96 and CFX96 Touch Systems do not require any passive
reference and the option is not available. For systems that have an option
for passive reference (for example, the Applied Biosystems 7500 Fast Dx
instrument), ensure that the passive reference option is switched off.

13  |  ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay

 Section 5 RT-PCR Procedure

2. Fluorescence Detectors (Dyes)

CFX96 or CFX96 Touch System

Target Fluorophore/Channel
ZIKV FAM
CHIKV HEX
DENV Texas Red
Internal control (IC) Cy5
RNase P (optional)* Cy5.5**
* To be bought separately; not included with the ZDC Multiplex RT-PCR Assay.
** T
 he RNase P assay is also available with TAMRA labeling. See Table 1 on page 9 for
system compatibility.

3. Temperature Profile and Dye Acquisition

Stage Cycle Repeats Acquisition Temperature, °C Time

Reverse
Transcription Hold 1 – 50 15 min
Denaturation Hold 1 – 94 2 min
Amplification Cycling 45 – 94 15 sec
– 55 40 sec
• 68 30 sec

ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay  |  14

Section 6 Interpretation of Results

Section 6
Interpretation of Results
■■  ll test controls should be examined prior to interpretation of sample
A
results. Table 3 provides a summary of the different recommended controls
as well as their expected results
■■  Cq value will be assigned for each amplification reaction occurring in a
A
reaction well. The Cq value represents the cycle at which the fluorescence
detected for a specific channel exceeds the set threshold. The Cq resulting
from interpolation of the fluorescence curve and the threshold can be the
result of fluorescence generated by a specific amplification of the desired
target or it can be a nonspecific fluorescence signal
■■  mplification plots should be analyzed for all samples. A positive
A
amplification is represented by an exponential increase in fluorescence
over the background. If the raw data (baseline not subtracted/normalized)
does not exhibit any exponential increase of fluorescence, the target has
probably not been amplified. If the amplification plot does not exhibit an
exponential fluorescence increase crossing the threshold, or if the baseline-
not-subtracted plot of Bio-Rad's CFX96™ or CFX96 Touch™ Real-Time
PCR Detection System does not exhibit an increase in fluorescence, the
target molecule has probably not been amplified
■■ If the analysis software assigns a Cq value to the amplification curve but
the amplification curve does not exhibit a sustained exponential growth
(followed by a plateau) that crosses the set threshold, the sample should
be considered undetermined
■■ An internal control should be included in every sample
■■  q values for the internal control and Zika (ZIKV), dengue (DENV), and
C
chikungunya (CHIKV) viruses could vary depending on sample type and
quality. Proper experimental procedure should be followed to accurately
define the correct setup conditions for different specimens

15  |  ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay

 Section 7 Assay Limitations

Test Assessment
Table 3. Test validity determination — summary of expected results.
Control Control Internal
Type Name Purpose of Control ZIKV DENV CHIKV Control
ZDC ZIKV + Quality of the extraction
Positive DENV + procedure; environmental
Control CHIKV + contaminants; determination + + + +
IC of primers/probes
malfunction; RT
No Water Environmental contaminants;
Template primers/probes — — — —
Control malfunctioning
Negative ZIKV + Quality of the extraction
Control DENV + procedure; environmental
CHIKV + contaminants; determination — — — +
IC of primers/probes
malfunction

For a test to be reliable, the controls should meet the following criteria:
 he negative control must be negative, showing no fluorescent signal
a. T
above the threshold in the ZIKV (FAM), DENV (Texas Red), and CHIKV
(HEX) channels. Cq cut-off values for the internal control (Cy5) should be
determined by the operator based on experimental design.

 he positive control must be positive, showing a fluorescent signal in

b. T
the ZIKV (FAM), DENV (Texas Red), CHIKV (HEX), and internal control (Cy5)
channels. Cq cut-off values should be determined by the operator based
on experimental design.

c. T
 he internal control must be positive for all samples, the positive
control, and the negative control (except NTC). Cq cut-off values for
the internal control (Cy5) should be determined by the operator based on
experimental design.

Section 7
Assay Limitations
This test is intended for research use only and not for use in diagnostic
procedures.

ZDC (Zika, Dengue, and Chikungunya) Multiplex RT-PCR Assay  |  16

 Applied Biosystems is a trademark of Thermo Fisher Scientific. Cy is a
trademark of GE Healthcare. FAM, HEX, QuantStudio, ROX, TAMRA, and
Texas Red are trademarks of Life Technologies Corporation. LightCycler
is a trademark of Roche Diagnostics. Rotor-Gene is a trademark of
the QIAGEN Group. Wescodyne is a trademark of American Sterilizer
Company Corporation.

Bio-Rad’s thermal cyclers and real-time thermal cyclers are covered by one
or more of the following U.S. patents or their foreign counterparts owned
by Eppendorf AG: U.S. Patent Numbers 6,767,512 and 7,074,367.

Bio-Rad
Laboratories, Inc.

Life Science Web site bio-rad.com USA 1 800 424 6723 Australia 61 2 9914 2800
Austria 43 01 877 89019 Belgium 32 03 710 53 00 Brazil 55 11 3065 7550
Group Canada 1 905 364 3435 China 86 21 6169 8500
Czech Republic 36 01 459 6192 Denmark 45 04 452 10 00
Finland 35 08 980 422 00 France 33 01 479 593 00
Germany 49 089 3188 4393 Hong Kong 852 2789 3300
Hungary 36 01 459 6190 India 91 124 4029300 Israel 972 03 963 6050
Italy 39 02 49486600 Japan 81 3 6361 7000 Korea 82 2 3473 4460
Mexico 52 555 488 7670 The Netherlands 31 0 318 540 666
New Zealand 64 9 415 2280 Norway 47 0 233 841 30 Poland 36 01 459 6191
Portugal 351 21 4727717 Russia 7 495 721 14 04 Singapore 65 6415 3188
South Africa 36 01 459 6193 Spain 34 091 49 06 580
Sweden 46 08 555 127 00 Switzerland 41 0617 17 9555
Taiwan 886 2 2578 7189 Thailand 66 2 651 8311
United Arab Emirates 971 4 8187300 United Kingdom 44 01923 47 1301

10000071135 Ver B (12004603) US/EG 18-0157 0118 Sig 0118